-->PONE-D-25-52195-->-->Can dietary supplementation a highly purified fucoidan alter growth, digestive enzyme activity, serum biochemicals, and immune-antioxidant response and related gene expressions of Nile tilapia?-->-->PLOS ONE

Dear Dr. Hassaan,

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PLOS ONE

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4. Please amend your authorship list in your manuscript file to include author Mohamed Hassaan, A. Elashry.

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6. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This manuscript investigates fucoidan supplementation effects on Nile tilapia growth, physiology, and immune responses. While the topic is relevant to aquaculture, the manuscript requires substantial revisions before publication consideration.

My concerns:

1. Sample Size Ambiguity:

-Lines 100-101 state fish were "divided into three groups with three replicates" but Table 3 and throughout show FIVE treatment groups (Control + 4 fucoidan levels)

-This fundamental inconsistency raises serious questions about the actual experimental design

QUESTION: Was this a 3-group or 5-group study? How many total tanks were used?

Stocking Density Concerns:

-20 fish in 150L tanks = 7.5L per fish initially, but final weights of 30g would make this extremely crowded

-No water quality data provided despite claim parameters were "maintained within recommended range"

-Could Provide actual water quality measurements (DO, ammonia, nitrite, pH, temperature) if possible

2. Methodological Gaps

Fucoidan Characterization Missing:

No molecular weight, purity, or sulfate content data for the fucoidan used

These properties critically affect bioactivity

Dose Selection Not Justified:

Why were 0.5, 1.0, 1.5, 2.0 mg/kg chosen?

Previous studies cited used 1% (10,000 mg/kg) - your doses are 5,000-10,000× lower

QUESTION: Are these doses realistic? Is there a unit error (should it be g/kg not mg/kg)?

Sampling Protocols Unclear:

"Three fish from each tank" for histology but "four fish" for enzyme analysis (line 120)

Were these same or different fish?

24-hour fasting before sampling not mentioned

3. Results Interpretation Problems

Biological Significance vs. Statistical Significance:

Many "significant" differences are trivially small

Example: WBC count 32.21 vs 45.38 (×10⁴/mm³) - is this biologically meaningful?

Growth differences of 1-2g between adjacent treatments may not be economically significant

Inconsistent Dose-Response:

Some parameters show linear increase (growth), others plateau (survival)

No discussion of optimal dose determination

Claims of "best" performance at 2.0 mg/kg lack statistical support (often not different from 1.5 mg/kg)

QUESTIONS:

-Feed Manufacturing: How was fucoidan mixed into feeds? Was stability verified? Storage conditions?

-Intestinal Morphometry (Table 4): How many villi measured per fish? Were measurements blinded?

-Enzyme Assays (Table 5): Were samples run in duplicate/triplicate? What were intra-assay CVs?

-Hematology (Table 6): Manual or automated counting? Differential count method?

-Gene Expression: Why were these three genes selected? What about IL-10, TNF-α, other immunity genes?

-Challenge Test: Why was no pathogen challenge performed to validate immunostimulant claims?

Some minor comments

Abstract & Introduction

Line 28: "Fish weighing 2.51±0.12" - missing units

Line 51: "water pollution with a negative impact of human activities" - awkward phrasing

Line 68: "Fucoidan is a natural polysaccharide" - needs "found in" or "extracted from"

Materials & Methods

Line 108: "A basal diet (310 g kg⁻¹ crude protein)" - inconsistent with Table 1 showing 316.35g/kg

Discussion

Line 254: "however, 2 mg kg⁻¹ diet fucoidan displayed the highest" - sentence fragment

Line 274: "Ozório et al. 2015" - inconsistent citation format (should be in parentheses)

Line 349: "IgM" appears without definition earlier in text

Some literatures should be used to enhance this manuscript:

-https://www.sciencedirect.com/science/article/abs/pii/S0377840125001130

-https://link.springer.com/article/10.1007/s10499-025-01960-7

-https://link.springer.com/article/10.1007/s10499-025-02205-3

Reviewer #2: The study is well written with obvious results..

The following adjustments are suggested during the revision:

-Abstract section have to cover your goal of this manuscript rewrite this section

-Abstract section is too long also compare treatments with p value in this section

-In introduction section, add more information about role immunostimulatns as feed addative on aquatics animals pleaseconsider below publications:

Benefits of Dietary Polyphenols and Polyphenol-Rich Additives to Aquatic Animal Health: An Overview

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa

Can dietary ginger (Zingiber officinale) alter biochemical and immunological parameters and gene expression related to growth, immunity and antioxidant system in zebrafish (Danio rerio)?

-Extract of grape seed enhances the growth performance, humoral and mucosal immunity, and resistance of common carp (Cyprinus carpio) against Aeromonas hydrophila

Cornelian cherry (Cornus mas L.) fruit extract improves growth performance, disease resistance, and serum immune-and antioxidant-related gene expression of common carp (Cyprinus carpio)

Effects of dietary Plantago ovata seed extract administration on growth performance and immune function of common carp (Cyprinus carpio) fingerling exposed to ammonia toxicity

-In line 109, according to which refrences select these dose for incorporation?

-In table 2, write name of gene as completely

-This manuscript need to be revise as English grammer

-

Reviewer #3: Plos One

Manuscript Title: Can dietary supplementation a highly purified fucoidan alter growth, digestive enzyme activity, serum biochemicals, and immune-antioxidant response and related gene expressions of Nile tilapia?

Manuscript Number: PONE-D-25-52195

General comment: Empirical data strongly supports the manuscript, making a unique contribution. Before publication, minor changes are necessary that should be made are shown below.

1)Please revise the title to be “Can dietary supplementation with highly purified fucoidan alter growth, digestive enzyme activity, serum biochemicals, immune-antioxidant responses, and related gene expressions in Nile tilapia (Oreochromis niloticus)?”

2)Line 28: Add the replicate number and the total number of fish used in the experiment.

3)Line 35: Replace “versus to basal diet” with “compared to the basal diet.”

4)Line 43: Please use keywords different from the title. For instance, you may use: immune response, gene expression, growth performance, antioxidant enzymes, dietary supplementation.

5)The first paragraph of the introduction contains several grammatical and structural issues. Please revise. For example Lines 50-51: “Shifting from extensive to intensive aquaculture systems increases vulnerability to pathogen infections, water pollution, and stress.”

6)The introduction heavily relies on outdated literature. Most references are over a decade old. The manuscript would benefit from a comprehensive update using recent, relevant research from the past five years as possible.

7)Please italicize all scientific names of species (e.g., P. monodon).

8)Line 97: Please specify the scientific name of Nile tilapia (Oreochromis niloticus) the first time it appears in this section.

9)Clarify the source of the fish (e.g., local hatchery, supplier name, or region).

10)Line 101: The Abstract mentions five groups, while this section states “three groups.” Please verify and correct.

11)Line 106: Add water quality parameters (temperature, pH, dissolved oxygen, ammonia, etc.) and the range maintained during the experiment.

12)Table 1: The calculation of Nitrogen-Free Extract is incorrect; please revise it and then adjust the Gross Energy accordingly.

13)Throughout the manuscript, fish gene names should be written in lowercase italics (e.g., il-1β, ifn-γ, hsp70), while protein names should appear in uppercase, non-italicized font (e.g., IL-1β, IFN-γ, HSP70). Please revise the manuscript accordingly to maintain proper scientific formatting and consistency.

14)Table 5: The significance letters indicating statistical differences should be written as superscripts. Please revise the table accordingly to ensure proper formatting.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Can dietary supplementation with highly purified fucoidan alter growth, digestive enzyme activity, serum biochemicals, immune-antioxidant responses, and related gene expressions in Nile tilapia (Oreochromis niloticus)?

PONE-D-25-52195R1

Dear Dr. Hassaan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Lee Seong

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: All comments have been addressed adequately by the authors. Accordingly I accept it for publication.Good luck

Reviewer #2: Dear Author

This revised version in good and acceptable, response requested comments point by point.

Reviewer #3: The authors adequately responded to all comments and performed all required modifications.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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